Wolbachia infection in native populations of Blattella germanica and Periplaneta americana

Cockroaches are significant pests worldwide, being important in medical, veterinary, and public health fields. Control of cockroaches is difficult because they have robust reproductive ability and high adaptability and are resistant to many insecticides. Wolbachia is an endosymbiont bacterium that infects the reproductive organs of approximately 70% of insect species and has become a promising biological agent for controlling insect pests. However, limited data on the presence or strain typing of Wolbachia in cockroaches are available. PCR amplification and sequencing of the wsp and gltA genes were used to study the presence, prevalence and molecular typing of Wolbachia in two main cockroach species, Blattella germanica (German cockroach) and Periplaneta americana (American cockroach), from different geographical locations of Iran. The Wolbachia endosymbiont was found only in 20.6% of German cockroaches while it was absent in American cockroach samples. Blast search and phylogenetic analysis revealed that the Wolbachia strain found in the German cockroach belongs to Wolbachia supergroup F. Further studies should investigate the symbiotic role of Wolbachia in cockroaches and determine whether lack of Wolbachia infection may increase this insect’s ability to tolerate or acquire various pathogens. Results of our study provide a foundation for continued work on interactions between cockroaches, bacterial endosymbionts, and pathogens.


Introduction
Cockroaches have survived on Earth for more than 300 million years, virtually unchanged. They are one of the most successful groups of animals because of their adaptability to various environmental conditions [1]. There are about 4500 species of cockroaches in the world, among which about 30 species frequently cohabit with human populations, and a few are indoor pests that are important in medical, veterinary, and public health fields [2,3]. The indoor health pests prefer humid, dark, and dirty environments, where they are exposed to various microorganisms [4][5][6]. Health, Tehran University of Medical Sciences. The cockroaches were identified using relevant taxonomic keys and descriptions [39]. A subset of individuals from each species and location was selected and preserved at -80˚C for subsequent molecular investigation.

Tissue dissection
The legs and wings of specimens were removed before isolating the gut and reproductive tissues. Tissue-specific dissection was carried out on each sample and dissected tissues, including the reproductive organs and alimentary canal, of each specimen were individually placed into small Petri dish in diameter size of 35 mm on ice to prevent DNA degradation. All dissection equipment and microscope slides were thoroughly wiped with 70% ethanol before commencing dissection of each sample.

DNA extraction and PCR amplification
DNA was extracted from each specimen using a QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, and the extracted DNA was stored at minus 20˚C. In this study, two molecular markers, Wolbachia surface protein (wsp) and the citrate synthase (gltA) genes, were used to detect Wolbachia infection. The wsp general primers were 81F (5 0 -TGGTCCAATAAGTGATGAAGAAAC-3 0 ) and 691R (5 0 -AAAAATTAAACGCTACTC CA-3 0 ) [40] and the gltA primers, were WgltAF1 (5 0 -TACGATCCAGGGTTTGTTTCTAC-3 0 ) and WgltARev2 (5 0 -CATTTCATACCACTGGGCAA-3 0 ) [41] and these served to confirm Wolbachia identity. For the wsp gene amplification, extracted DNA samples from the dissected tissues were screened by PCR in a thermocycler (Eppendorf, Hamburg, Germany) using the following protocol: initial denaturation at 95˚C for 4 min; 35 cycles of 94˚C 1min, 55˚C 1min and 72˚C 1min and final elongation step of 72˚C for 10 min. For the gltA gene amplification, initial denaturation at 95˚C for 2 min; two cycles of denaturation at 95˚C for 2 min, annealing at 60˚C for 1 min and extension at 72˚C for 1 min; 35 cycles of denaturation at 95˚C for 30s, annealing at 60˚C for 1 min and extension at 72˚C for 45s; final extension at 72˚C for 10 min.
All PCR procedures were performed in reaction mixtures consisting of 12.5μl of Taq DNA Polymerase Master Mix RED (Denmark), 3μl of extracted DNA, and 1μl each of 5μM forward and reverse primers for Wolbachia PCR screens. Double-distilled water was used to top up the reaction mixture to a final volume of 25μl. PCR amplification of positive and negative controls was also conducted simultaneously. The negative controls were prepared with ddH 2 O and DNA from a male Anopheles stephensi mosquito; a positive control was prepared in our laboratory using DNA extracted from Drosophila melanogaster which harbours Wolbachia wMelPop strain. Amplicons were separated by gel electrophoresis on 1.5% agarose gel stained with green viewer (Parstous, Iran) and visualised under an ultraviolet transilluminator.

Gene sequencing and phylogenetic analysis
The criteria set to confirm Wolbachia infection were based on successful amplification of the molecular markers. Furthermore, samples that met this criterion were sequenced by bidirectional sequencing at Genomin Sequencing Centre, Iran. A subset of the PCR products of wsp and gltA gene representatives of different locations were purified from gels using a gel purification kit and subjected to sequencing. Sequencing was performed using an ABI 3730 sequencer machine. The resultant sequences were checked to correct ambiguities. Homologies with the available sequence data in GenBank were checked by using basic local alignment search tool (BLAST) analysis software (www.ncbi.nlm.nih.gov/BLAST). A subset of consensus sequences for both loci was deposited in the GenBank database (Table 2). A subset of the available representative Wolbachia wsp and gltA sequences of nine supergroups (A-G, M, and T for wsp gene and A-F, and H-K for gltA gene) were acquired from public databases (http://www.ncbi.nlm) for phylogenetic analysis (Table 3). Owing to the different lengths of these sequences, all those used for alignment were trimmed to obtain consistent regions that were 443 bp for wsp and 599 bp for gltA genes, respectively. Multiple alignments of the sequences were carried out using the Clustal W algorithm in software MEGA X [42]. The short sequence reads were excluded. Pairwise sequence divergence, using Kimura's 2-parameter distance algorithm, and neighbour joining (NJ) tree, shown in Figs 2 and 3, were processed in MEGA X. The robustness of all phylogenetic trees was tested with a bootstrapping value in NJ. The wsp sequence of Anaplasma centrale (Genbank ID: AB211162) and gltA sequence of Bartonella quintana (Genbank ID number: M73228) were acquired from Genbank and used as outgroups for each gene ( Table 2).

Data analysis
Statistical significance was determined as P < 0.05. All statistical analyses were performed in SPSS statistics version 21.

Wolbachia detection and prevalence
A total of 965 cockroaches, representing two species, were collected from 13 localities in Iran (Fig 1). PCR assays, using the primers described in Materials and Methods, gave the expected

PLOS ONE
amplification products of 632 bp and 659 bp, for wsp and gltA respectively. Overall, only B. germanica was found to host Wolbachia. The other cockroach species (P. americana) showed no PCR amplification products. From a total of 544 German cockroaches screened by wsp marker, 95 specimens (17.46%) were found to be infected with Wolbachia (Table 3). The wspnegative samples were re-evaluated with the gltA gene primers, and 17 out of 449 wsp-negative samples were found to be gltA-positive; thus, the totals number of Wolbachia-infected German cockroaches was 112 out of 544 (20.6%). The prevalence of Wolbachia infection was almost twice as high in females as in males (59 versus 32), which is statistically significant (P<0.001). The Blast search of the wsp and gltA sequences of infected B. germanica revealed a high homology, with the F supergroup of Wolbachia strains found in Cimex lectularius (AP013028), Blattella sp (DQ354917), and Supella longipalpa (EF193198) (Figs 2 and 3). Following Vaishampayan et al's finding, the wsp sequences obtained in this study can be classified into Wolbachia F supergroup [36]. According to our knowledge, this is the first report of Wolbachia infection in B. germanica in Iran (Table 3).

PLOS ONE
The sequences generated during this study have been deposited in the Gen Bank database (OM928504-OM928515 for wsp and OP146461-OP146467 for gltA sequences).

Phylogenetic analysis
The neighbour joining phylogenetic tree between the Wolbachia strain identified in this study and other known available Wolbachia strains are shown in Figs 2 and 3. Fig 2 shows a phylogenetic tree inferred from 433 bp of wsp gene sequences from Iranian isolates of B. germanica and some representative sequences of Wolbachia strains belonging to supergroups A, B, C, D, E, F, G, and M obtained from Genbank database. The phylogenetic analysis showed that Wolbachia strains identified from the Iranian specimens were closely associated and clustered with the Wolbachia strains presented in Blattella sp (DQ354917) and Supella longipalpa (EF193198) of supergroup F (Fig 2). In addition, in the phylogenetic analysis of gltA sequences, independent of the method for tree reconstruction, the cockroach Wolbachia sequences from the Iranian German cockroaches clustered with supergroup F (Fig 3) and led to similar tree topologies as found with the wsp gene. Phylogenetic analysis of the Wolbachia sequences with Maximum likelihood (ML), Neighbour Joining (NJ) and Maximum Parsimony (MP) methods showed almost similar topology.

Discussion
In the current research we found that only about twenty percent of B. germanica specimens collected from the 13 provinces of Iran were positive for Wolbachia. This result concurs with the study of Vaishampayan et al in India, which reported that 20% of B. germanica examined harboured Wolbachia [38]. Also, we found no Wolbachia infection in American cockroaches. The low rate of Wolbachia infection in the German cockroaches and the lack of infection in the American cockroaches may indicate a lack of dependence, or a very low dependence, of the cockroach species on the endosymbiont for their survival and reproduction, in what is known as obligatory relationships [27, 56, 57]. Therefore, the low level of Wolbachia infection in cockroaches negates the possibility of Wolbachia being an obligatory endosymbiont in these cockroach species. Also, it is possibility that Wolbachia may have some negative effects on the fitness of cockroaches, which would counteract the possibility of Wolbachia expanding in the population through providing fitness advantages.
The low rate or lack of Wolbachia infection in the cockroaches may change in future because it is shown that changing the gut microbiota composition with antibiotic treatment enhanced Wolbachia density in Drosophila melanogaster [58]. We have no evidence to assess the impact of antibiotic treatment on the incidence and frequency of Wolbachia in German cockroaches, however, cockroaches are exposed to antibiotics in places such hospitals [59, 60] and most bacterial agents isolated from cockroaches are antidrug-resistant and antibioticresistant [61]. These situations provide cockroaches with diverse antibiotic treatments which may result in raising Wolbachia density in future and could be the subject of future studies. The Wolbachia strain found in German cockroaches in this study belongs to supergroup F, which is consistent with previous studies indicating supergroup F in other cockroaches, such as Supella longipalpa and Blattella sp [38]. Wolbachia supergroup F has also been detected in bedbugs and nematodes. The Wolbachia supergroup F is essential for the bedbugs' growth and reproduction because the bacterium provides B vitamins, which are deficient in their bloodbased diet [27]. Therefore, the Wolbachia strain might promote persistence by providing fitness advantages to the German cockroach via nutrient supplementation [27,62]. However, we do not yet know if this strain could provide any nutrient supplement for German cockroaches, and this needs to be determined by further studies.
Wolbachia strains are divided into 20 supergroups, ranging from A to U (G was not considered anymore) which diverged around 100 million years ago, first in filarial nematodes and then infecting arthropods [63]. In this study we found F supergroup in the cockroaches which is also found in distantly related host species including nematodes and domestic indoor pests (Cimex, Supella and Blattella). Wolbachia is transmitted either vertically between host generations or horizontally to other individuals and species through a mechanism called host shift (HS) [63][64][65].
German or American cockroaches are omnivorous synanthropic insects, frequently encountering high loads of diverse microbes, and are reservoirs and vectors of several pathogens particularly pathogenic bacteria [61,[66][67][68]. For example, Dokor showed that about a quarter of the microorganisms isolated from cockroaches are food-borne pathogens including Escherichia coli O157:H7, Staphylococcus aureus, Bacillus cereus, Shigella dysenteriae, Salmonella enterica subsp. enterica serovar Typhi, Rotavirus, Aspergillus fumigatus, and Cryptosporidium parvum [66]. Although Wolbachia has been shown to protect insects from a range of microbial and eukaryotic pathogens including viruses, Plasmodium and filarial nematodes [31][32][33][34][35][36][37], there is no strong evidence that Wolbachia-infected insects can be protected against pathogenic bacteria. In an experiment, no difference in mortality was observed in the Drosophila simulans lines with five different Wolbachia strains or without Wolbachia when the lines were challenged with the pathogenic bacteria. Similarly, no antibacterial protection or upregulation of the antibacterial immune genes was observed for D. melanogaster infected with Wolbachia compared to paired flies without Wolbachia. It was suggested that Wolbachia-mediated antibacterial protection is not universal in insects and furthermore that the mechanisms of antibacterial and antiviral protection are independent [69]. In another study it was found that D. melanogaster flies harbouring no endosymbionts, those carrying both Spiroplasma and Wolbachia, and those containing Wolbachia only had parallel survival rates following infection with the virulent insect pathogen Photorhabdus luminescens and non-pathogenic Escherichia coli bacteria [70]. Also, Wolbachia presence did not provide a protective advantage against entomopathogenic fungi, Beauveria bassiana and B. brongniartii, in two important mosquito vectors, Aedes albopictus and Culex pipiens that naturally carry Wolbachia [71]. Taken together these results plus high microbial loads, we suggest that presence of Wolbachia supergroup F may not provide protection in the German cockroach species. Also, having found Wolbachia supergroup F in German cockroaches warrants further studies to determine if Wolbachia supergroup F can manipulate the cockroach's reproduction system through such means as cytoplasmic incompatibility (CI), induction of parthenogenesis (IP), male-killing (MK), or feminization of genetic males (MF) [46, [72][73][74].
In this study the cockroaches were molecularly screened for Wolbachia DNA using two primer sets targeting partial wsp and gltA genes. Comparing the results of this study pointed the discrepancy in results between the primer pairs where Wolbachia DNA detected at the wsp locus was less than at the gltA locus. Baldo et al [75] showed that wsp gene has a mosaic structure with four hypervariable regions (HVRs), which provide a reasonable explanation for the negative results. It is possible that there is a trade-off between sensitivity and specificity of primer sets and certain primer sets can be more efficient than others, but that no single protocol can ensure the specific detection of all known Wolbachia infections [76].
In the present study we found that there was a sex bias toward infection in females of B. germanica, with Wolbachia prevalence in females being higher than in males. Wolbachia infections tend to confer reproductive advantages on their female hosts, which is the sex responsible for vertical transmission of the bacteria from one generation to another, leading to increased prevalence and propagation within the host populations [74]. Higher Wolbachia infection in females has already been reported in few hosts, such as hard ticks [77], fruit flies [78], and fleas [79]. Low levels of Wolbachia infection in male cockroaches also tends to support rejection of the possibility of Wolbachia as an obligatory endosymbiont, at least in males. Finally, the low occurrence of Wolbachia in male cockroaches may suggests the absence of robust CI in German cockroaches.

Conclusion
In conclusion, we found low or no Wolbachia infection in German and American cockroaches, respectively, calling for additional surveys of hidden fitness, nutrition, or protection properties, the reproductive manipulation such as CI occurrence, and underlying systems with sex-bias differences in Wolbachia persistence. Nevertheless, the long evolutionary history of Wolbachia's interaction with invertebrate hosts and its adaptations for germ line transmission contribute to the value of Wolbachia for control of insect pests.